The overall goal of the proposed research is to study and establish the molecular basis for thee substrate specificity of mixed-function oxidase systems in hepatic microsomes, which are known to hydroxylate fatty acids and steroids as well as drugs, hydrocarbons, carcinogens, and other foreign compounds. The two major forms of rabbit liver microsomal cytochrome P-450, phenobarbital-inducible P-450LM2 and beta-naphthoflavone-inducible P-450LM4, as well as NADPH-cytochrome P-450 reductase and cytochrome b5 wiill be purified to homogeneity and their interactions with several mixed function oxidase substrates will be thoroughly characterized by 1H, 13C and 31p Fourier transform nuclear magnetic resonance spectroscopy and UV-visible difference spectrophotometry. The structural features of substrate binding and orientation in proximity to the catalytic site of cytochrome P-450 will be ascertained by the use of NMR T1 relaxation time measurements and split beam spectrophotometry. Similar techniques will be employed to examine the effect of NADPH-cytochrome PP-450 reductase, phospholipid, and cytochrome b5 (added alone or together in stepwise fashion) on the previously characterized substrate-cytochrome P-450 interactions. The role of different fatty acid residues in various phospholipids on the interaction of substrates with P-450LM2 and P-450LM4 will also be evaluated. Information on the substrate binding cavity, and thus substrate accessibility to the hemeprotein catalytic site, will be obtained from variable temperature relaxation rate studies of substrate-cytochrome P-450 interactions in the absence and presence of other mixed-function oxidase components and related enzymes. Spectral dissociation constants (KD) will be used in conjunction with 1H and 13C relaxation rate changes to determine distances of approach of the individual molecular sites to the iron atom in the heme prosthetic group of the cytochrome. The catalytic activities of P-450LM2 and P-450LM4 toward the various compounds employed in the NMR and spectral studies will be routinely examined by reconstituting the cytochrome with the purified NADPH-cytochrome PP-450 reductase and phospholipid in the presence of NADPH and oxygen.